Wilson D H, Benight A S
Department of Chemistry, University of Illinois, Chicago 60680.
J Biol Chem. 1990 May 5;265(13):7351-9.
Total intensity light scattering is employed to investigate the self-assembly kinetics of RecA protein. Reaction conditions are employed where the kinetics of self-assembly are slow enough to yield reliable scattered intensity measurements over the range of scattering angles from 40 to 130 degrees as a function of time. From these measurements the time-dependent behavior of the weight average molecular weight, Mr, and radius of gyration, RG, of the associating protein species as a function of [MgCl2], [NaCl], [RecA], and pH was determined. The temperature dependence of RecA self-assembly was also investigated and allowed an evaluation of the activation thermodynamic parameters of association. Results reveal RecA self-assembly is bi-phasic under all conditions examined. The first phase, referred to as "filamentation" is second-order in [RecA] and occurs via a quasi linear condensation scheme with an Arrhenius activation energy of 88.6 kcal/mol. Filamentation assembly involves the uptake of one proton, one MgCl2, the release of five to six NaCls, and is driven by the release of approximately 70 water molecules. The evaluated activation parameters of the first kinetic phase are consistent with the proposition that linear self-assembly of RecA protein into ordered filaments is entropically driven. The second kinetic phase, referred to as "bundling" is greater than second-order in both [RecA] and [MgCl2], is considerably slower that filamentation assembly, and is apparently initiated by 2nd order collisions of linear filaments.
利用总强度光散射来研究RecA蛋白的自组装动力学。所采用的反应条件是，自组装动力学足够缓慢，以便在40至130度的散射角范围内作为时间的函数获得可靠的散射强度测量值。通过这些测量，确定了缔合蛋白物种的重均分子量Mr和回转半径RG随时间的变化行为，以及它们与[MgCl2]、[NaCl]、[RecA]和pH的关系。还研究了RecA自组装的温度依赖性，并对缔合的活化热力学参数进行了评估。结果表明，在所有检测条件下，RecA自组装都是双相的。第一阶段，称为“丝状化”，对[RecA]呈二级反应，通过准线性缩合机制发生，阿累尼乌斯活化能为88.6千卡/摩尔。丝状化组装涉及摄取一个质子、一个MgCl2，释放五到六个NaCl，并由大约70个水分子的释放驱动。第一动力学阶段的评估活化参数与RecA蛋白线性自组装成有序丝状结构是由熵驱动的这一观点一致。第二动力学阶段，称为“成束”，对[RecA]和[MgCl2]均大于二级反应，比丝状化组装慢得多，显然是由线性丝的二级碰撞引发的。
